The microphthalmia (mi) gene is critical for osteoclast development based on severe osteoclast defects in mi/mi mutant mice. These mice represent a genetically defined animal model of osteopetrosis secondary to profound failure of osteoclast function, despite normal osteoclast numbers. Osteoclasts play an important role in the pathogenesis of osteoporosis, osteopetrosis, and a variety of pathological features of metastatic cancer, such as bony invasion, pathologic fractures, and bone pain caused by many human tumors. Thus, osteoclast function lies at the crossroads of many human diseases of particular relevance in bone development and aging. In addition, osteoclasts provide an attractive system to study the function of the transcription factor microphthalmia (Mi). The mi gene encodes a basic/helix-loop-helix/leucine zipper (bHLH-ZIP) transcription factor related to the oncoprotein, Myc. The mi mutant phenotype in mice includes osteopetrosis, a lack of pigmentation, small eyes, and a mast cell defects. Our laboratory has biochemically characterized DNA binding, transcriptional activity, and identified three dimerization partners of Mi. In addition we have recently discovered that the Mi protein is phosphorylated in response to c-kit (stem cell factor receptor) activation via a signaling pathway involving MAP kinase and that this phosphorylation enhances Mi transcriptional activation. This observation was sparked by the similarity of pigment cell defects in mi/mi and kit mutant mice. Mice with mutations in M-CSF develop osteopetrosis. M-CSF receptor is closely related to c-kit, and in present preliminary evidence that when primary osteoclast-like cultures are stimulated with M-CSF, the Mi protein undergoes a post- translational modification (likely phosphorylation). Given that both M-CSF and Mi are critical for osteoclasts, our observation may relate these factors in osteoclast signaling and transcription. The overall goal of this project will be to elucidate the critical function of the Mi transcription factor in osteoclast development. The specific aims are 1) To Characterize Mi's expression and function during osteoclast development, 2) To examine the possible regulation of Mi via M-CSF receptor signaling and 3) To analyze potential genes transcriptionally regulated by Mi in osteoclasts. Mi expression and dimerization partners will be analyzed by immunohistochemistry, immunoprecipitation, and Western blot analysis with monoclonal and polyclonal antibodies that I have developed and characterized. Mi's potential role in M-CSF signaling will be assessed by analyzing the MAP kinase pathway and in vitro kinase assays using Mi or specific mutants as substrate. Variety of Mi expression constructs have been engineered including dominant negative mutants and will be employed to help identify target genes transcriptionally regulated by Mi in osteoclasts. Dr. David Fisher will supervise the project and head an advisory board of experts in signalling, bone biology, cellular physiology formed to provide additional guidance and aid in my transition to an independent investigator.